The heretical nature of prions and the reported transmission of the disease between animals to humans are important to fully understand key aspects of prion biology, including the biochemical and structural basis of species barrier, strain diversity, and the relationship between prion replication and neurodegeneration. A better understanding of these issues would contribute to prevent the emergence of new diseases with potential for epidemic consequences and to develop much needed therapeutic strategies for these incurable diseases. The main goal of this project is to elucidate the molecular basis of prion strains, their influence on species barrier and the biochemical/structural properties modulating the replication and pathogenicity of prions as well as use of this knowledge to develop a novel therapeutic strategy for prion diseases. The project is structured around several highly innovative hypotheses. In specific aim 1 we will test the hypothesis that that the animal to human species barrier depends on a complex interaction between the degree of similarity on the PrP primary sequence and the strain characteristics of the infectious agent. For these studies we will focus on the two most prevalent animal prion diseases: scrapie in sheep and CWD in cervids. In specific aim 2 we will test the hypothesis that the prion strain associated with vCJD in humans consists of an ensemble of PrPSc variants with distinct biochemical properties and that the tissue environment influences prion replication by selecting specific PrPSc conformations from the pool. These studies are inspired on our recent results indicating that the features of PrPSc accumulated in spleen or brain from vCJD patients are different. In specific aim 3 we propose to utilize the prion principle to generate a selfreplicating therapy for prion diseases by dissociating prion replication from pathogenicity. The plan is to generate PrPSc variants that efficiently replicates, but do not produce disease. This innocuous prion variant could outcompete a pathogenic prion by recruiting and converting the PrPC substrate into more of the therapeutic prion, preventing infectivity. Project Summary/